Mycelial cell wall fractionation was performed according to the method described by Fontaine et al.[86] (link) with slight modification. Briefly, wt and Δhac1 strains were grown in a 1.2-liter fermenter in liquid Sabouraud medium. After 24 h of cultivation (linear growth phase), the mycelia were collected by filtration, washed extensively with water and disrupted in a Dyno-mill (W. A. Bachofen AG, Basel, Switzerland) cell homogenizer using 0.5-mm glass beads at 4°C. The disrupted mycelial suspension was centrifuged (3,000×g for 10 min), and the cell wall fraction (pellet) obtained was washed three times with water, subsequently boiled in 50 mM Tris-HCl buffer (pH 7.5) containing 50 mM EDTA, 2% SDS and 40 mM β-mercaptoethanol (β-ME) for 15 min, twice. The sediment obtained after centrifugation (3,000×g, 10 min) was washed five times with water and then incubated in 1 M NaOH containing 0.5 M NaBH4 at 65°C for 1 h, twice. The insoluble pellet obtained upon centrifugation of this alkali treated sample (3,000×g, 10 min, AI-fraction) was washed with water to neutrality, while the supernatant (AS-fraction) was neutralized and dialyzed against water. Both fractions were freeze-dried and stored at −20°C until further use. Hexose composition in the samples were estimated by gas-liquid chromatography using a Perichrom PR2100 Instrument (Perichrom, Saulx-les-Chartreux, France) equipped with flame ionization detector (FID) and fused silica capillary column (30 m×0.32 mm id) filled with BP1, using meso-inositol as the internal standard. Derivatized hexoses (alditol acetates) were obtained after hydrolysis (4N trifluoroacetic acid/8N hydrochloric acid, 100°C, 4 h), reduction and peracetylation. Monosaccharide composition (percent) was calculated from the peak areas with respect to that of the internal standard.
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